Abstract Herpes simplex virus type 1 (HSV-1) is a significant human pathogen and is the leading cause of sporadic encephalitis in the USA. Strains of virus display virulence phenotypes ranging from avirulent to highly pathogenic. There are indications that the virulence of a given strain of virus is actually determined by the ?constellation? of genes carried by the virus and how they interact with each other; a phenomenon termed epistasis. Traditional methods for analyzing the function of viral genes are difficult to apply to studying epistatic interactions in virulence. Recently we described a novel approach, vQTLmap, that has allowed us to identify contributions of multiple genes to peripheral disease phenotypes. To date we have analyzed four quantitative phenotypes, blepharitis, stromal keratitis, corneal neovascularization, and weight loss. Surprisingly, there are distinct differences in the viral genes that appear to be driving each of these phenotypes. In order to carry out vQTLmap using quantitative measures of neurovirulence, we need to isolate additional neurovirulent recombinants between the OD4 and CJ994 parental viruses. The goals of this proposal are to generate the reagents (e.g., neurovirulent recombinants) needed to analyze neurovirulence genes driving HSV-1 encephalitis and identify epistatic interactions and carry out vQTLmap analysis of percent mortality, percent ataxia, percent head tilt, and percent limb paralysis. At the completion of this project, we will be able to submit an R01 application to carry out vQTLmap on features such as percent mortality, brain titers of virus, localization of the infection in the brain, and neuroinvasiveness. A better understanding of how viral genes work together to promote disease has the potential to identify novel targets that could assist antiviral drug or vaccine development efforts.